Galiakhmetova Diana, Koviarov Aleksandr, Dremin Viktor, Gric Tatjana, Stoliarov Dmitrii, Gorodetsky Andrei, Maimaris Marios, Shcherbakova Daria M, Baloban Mikhail, Verkhusha Vladislav V, Sokolovski Sergei G, Rafailov Edik
Aston Institute of Photonic Technologies, Aston University, Birmingham, UK.
Department of Electronic Systems, Vilnius Gediminas Technical University, Vilnius, Lithuania.
Protein Sci. 2025 May;34(5):e70118. doi: 10.1002/pro.70118.
Near-infrared (NIR) fluorescent proteins and optogenetic tools derived from bacterial phytochromes' photosensory core modules (PCMs) operate within the first (NIR-I) tissue transparency window under single-photon activation. Leveraging two-photon (2P) light in the second transparency window (NIR-II) for photoswitching bacterial phytochromes between Pr and Pfr absorption states offers significant advantages, including enhanced tissue penetration, spatial resolution, and signal-to-noise ratio. However, 2P photoconversion of bacterial phytochromes remains understudied. Here, we study the non-linear Pr to Pfr photoconversion's dependence on irradiation wavelength (1180-1360 nm) and energy fluence (41-339 mJ/cm) for the PCM of DrBphP bacterial phytochrome. Our findings reveal substantially higher photoconversion efficiency for the engineered monomeric DrBphP-PCM (73%) compared to the natural dimeric DrBphP-PCM (57%). Molecular mechanical calculations, based on experimentally determined 2P absorption cross-section coefficients for the monomer (167 GM) and dimer (170 GM), further verify these results. We demonstrate both short- (SWE) and long-wavelength excitation (LWE) fluorescence of the Soret band using 405 and 810-890 nm laser sources, respectively. Under LWE, fluorescence emission (724 nm) exhibits saturation at a peak power density of 1.5 GW/cm. For SWE, we observe linear degradation of fluorescence for both DrBphP-PCMs, decreasing by 32% as the temperature rises from 19 to 38°C. Conversely, under LWE, the monomeric DrBphP-PCM's brightness increases up to 182% (at 37°C), surpassing the dimeric form's fluorescence rise by 39%. These findings establish the monomeric DrBphP-PCM as a promising template for developing NIR imaging and optogenetic probes operating under the determined optimal parameters for its 2P photoconversion and LWE fluorescence.
近红外(NIR)荧光蛋白以及源自细菌光敏色素光感核心模块(PCM)的光遗传学工具,在单光子激活下于首个(NIR-I)组织透明窗口内发挥作用。利用第二透明窗口(NIR-II)中的双光子(2P)光,使细菌光敏色素在Pr和Pfr吸收态之间进行光开关转换,具有显著优势,包括增强组织穿透性、空间分辨率和信噪比。然而,细菌光敏色素的2P光转换仍未得到充分研究。在此,我们研究了DrBphP细菌光敏色素的PCM的非线性Pr到Pfr光转换对照射波长(1180 - 1360 nm)和能量通量(41 - 339 mJ/cm)的依赖性。我们的研究结果表明,与天然二聚体DrBphP-PCM(57%)相比,工程化单体DrBphP-PCM的光转换效率显著更高(73%)。基于实验测定的单体(167 GM)和二聚体(170 GM)的2P吸收截面系数进行的分子力学计算,进一步验证了这些结果。我们分别使用405和810 - 890 nm激光源展示了Soret带的短波激发(SWE)和长波激发(LWE)荧光。在LWE下,荧光发射(724 nm)在峰值功率密度为1.5 GW/cm时呈现饱和。对于SWE,我们观察到两种DrBphP-PCM的荧光均呈线性降解,随着温度从19°C升高到38°C,荧光强度下降32%。相反,在LWE下,单体DrBphP-PCM的亮度增加高达182%(在37°C时),比二聚体形式的荧光增强高出39%。这些发现确立了单体DrBphP-PCM作为一个有前景的模板,可用于开发在其2P光转换和LWE荧光的确定最佳参数下运行的近红外成像和光遗传学探针。
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